No description available.

No description available.

No description available.

The avian embryo is a popular animal model because it is widely available (Antin et al., 2004), it is easily manipulated, and it can provide important insights into normal and abnormal embryo development (Kulesa, 2004). While in vivo and in vitro cultures of chick embryos are common, in ovo cultures are rarer, and none have been designed where the egg did not have to be resealed afterwards. The present study aimed to develop a set-up in which the egg would be windowed without resealing the egg so that the embryo would remain accessible for experimental manipulation. As well, this study aimed to track cell movement during neurulation by microinjecting points of dye along the embryo. Two prototypes were developed based on the concept that temperature and moisture controlled air passing over the windowed egg would serve as a blanket. When these prototypes were unable to keep the embryo alive, a protocol developed by Kulesa and Fraser (2004) was adapted for the study. This protocol involved the construction of a Teflon window which was placed in the windowed egg and sealed with beeswax. Initial microinjection tests with Fast Green FCF showed that the dye dissipated quickly after injection, most likely because of the hydrophilicity of the dye. Therefore, a list of non-fluorescent, hydrophobic dyes were chosen and tested for suitability to cell tracking. Time restrictions prevented the actual cell tracking experiments from taking place, but it was found that Oil Red O fulfilled the criteria. As Oil Red O is usually used to identify lipids in static experiments, it remains to be seen whether it would function as a vital dye. Future experiments include expanding the set-up for use with a confocal microscope for a 4-D rendering of cell movement, and taking advantage of the symmetrical nature of neurulation in the chick embryo to examine perturbations to the normal progress of development, via drugs such as valproic acid.

The avian embryo is a popular animal model because it is widely available (Antin et al., 2004), it is easily manipulated, and it can provide important insights into normal and abnormal embryo development (Kulesa, 2004). While in vivo and in vitro cultures of chick embryos are common, in ovo cultures are rarer, and none have been designed where the egg did not have to be resealed afterwards. The present study aimed to develop a set-up in which the egg would be windowed without resealing the egg so that the embryo would remain accessible for experimental manipulation. As well, this study aimed to track cell movement during neurulation by microinjecting points of dye along the embryo. Two prototypes were developed based on the concept that temperature and moisture controlled air passing over the windowed egg would serve as a blanket. When these prototypes were unable to keep the embryo alive, a protocol developed by Kulesa and Fraser (2004) was adapted for the study. This protocol involved the construction of a Teflon window which was placed in the windowed egg and sealed with beeswax. Initial microinjection tests with Fast Green FCF showed that the dye dissipated quickly after injection, most likely because of the hydrophilicity of the dye. Therefore, a list of non-fluorescent, hydrophobic dyes were chosen and tested for suitability to cell tracking. Time restrictions prevented the actual cell tracking experiments from taking place, but it was found that Oil Red O fulfilled the criteria. As Oil Red O is usually used to identify lipids in static experiments, it remains to be seen whether it would function as a vital dye. Future experiments include expanding the set-up for use with a confocal microscope for a 4-D rendering of cell movement, and taking advantage of the symmetrical nature of neurulation in the chick embryo to examine perturbations to the normal progress of development, via drugs such as valproic acid.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [110]-127).

Many heavy metals are essential for biological functions; however some of these metals, especially at high concentrations, can have serious adverse effects on humans. The main sources of heavy metal exposure are through agriculture, transport, mining and related operations. South Africa has a thriving mining industry and is known for its rich mineral resources, but due to the incorrect method of disposal of the waste from these mines, substances, including heavy metals, get into the water and air supply, affecting the people living in close proximity to these mines. Exposure is through inhalation of contaminated air and consumption of contaminated food and water. The most vulnerable to heavy metals are the developing fetus, because of the high rate of cell division and differentiation. In the current study, two heavy metals cadmium (Cd) and chromium (Cr) were chosen based on the possibility of being exposed to them in South Africa. Thus, the aim of this study was to investigate the possible cellular effects of the heavy metals Cd and Cr alone and in combination, at different concentrations, on brain, liver and kidney tissue by using a chick embryo model. This model was successfully implemented over a 14 day period after which the embryos were terminated and the brains, livers and kidneys removed and processed for light- and transmission electron microscopy (with energy dispersive spectroscopy and electron energyloss spectroscopy). In addition, the effect of Cd and Cr alone and in combination on DNA structure and micronuclei formation was evaluated. The levels of the major antioxidant component, glutathione was determined in the brains of the chick embryos. At low concentration of Cd and Cr alone and in combination, a hormesis effect was observed in the survival rates and weights of the chick embryos, while at x1000 physiological dose (PD) Cr and Cd alone and in combination the effects were toxic. The majority of viable embryos did not have any macro-anatomy abnormalities. Morphological evaluation of the brain, liver and kidney samples revealed that Cd caused severe alterations at its highest concentration with minor alterations at the lower concentrations. Cr and the metal combination groups on the other hand, only caused minimal alterations throughout the concentration ranges evaluated. The presence of Cd and Cr alone and in combination in the liver tissue was confirmed with the electron energy-loss spectroscopy analysis that detected these metals in the nuclei, mitochondria and Golgi complexes of the hepatocytes. This might contribute to the ultrastructural changes observed in this organ. The genotoxicity testing on the red blood cells revealed no substantial differences, as only a few micronuclei were present. Although heavy metals cause DNA damage through an indirect mechanism of oxidative damage, the presence of Cd and Cr in the nucleus and mitochondria indicates that these metals may have a direct effect on DNA structure. With DNA agarose gel electrophoresis it was found that Cd and Cr alone and in combination caused DNA fragmentation. In the brain, GSH levels were normal; however changes may be the result of Cd and Cr causing the depletion of other antioxidant elements such as glutathione reductase, glutathione peroxidase, superoxide dismutase and catalase. In conclusion, this study indicates that Cd and Cr alone and in combination are toxic to the chick embryo. Cd is more toxic than Cr, and both metals accumulate in the nuclei and mitochondria where they induce damage either through oxidative and/or other mechanisms associated directly with DNA damage. / Dissertation (MSc)--University of Pretoria, 2014. / tm2015 / Anatomy / MSc / Unrestricted

Cannabinoids (CBs) are compounds that activate the CB1 and CB2 receptors. CB receptors mediate many different physiological functions, and cannabinoids have been reported to decrease tumor cell viability, proliferation, migration, as well as to modulate metastasis. In this thesis, the effects of cannabinoids on human colorectal carcinoma Caco-2 cells (Paper I) and mouse P19 embryonal carcinoma (EC) cells (Paper III) were studied.  In both cell lines, the compounds examined produced a concentration- and time-dependent decrease in cell viability. In Caco-2-cells, HU 210 and the pyrimidine antagonist 5-fluorouracil produced synergistic effects upon cell viability. The mechanisms behind the cytocidal effects of cannabinoids appear to be mediated by other than solely the CB receptor, and a common mechanism in Caco-2 and P19 EC cells was oxidative stress. However, in P19 EC cells the CB receptors contribute to the cytocidal effects possibly via ceramide production. In paper II, the association between CB1 receptor immunoreactivity (CB1IR) and different histopathological variables and disease-specific survival of colorectal cancer (CRC) was investigated. In microsatellite stable (MSS) cases there was a significant positive association of the tumor grade with the CB1IR intensity. A high CB1IR is indicative of a poorer prognosis in MSS with stage II CRC patients. Paper IV focused on the cytotoxic effects of cannabinoids during neuronal differentiation. HU 210 affected the cell viability, neurite formation and produced a decreased intracellular AChE activity. The effects of cannabinoids on embryonic development and survival were examined in Paper V, by repeated injection of cannabinoids in fertilized chicken eggs. After 10 days of incubation, HU 210 and cannabidiol (without CB receptor affinity), decreased the viability of chick embryos, in a manner that could be blocked by α-tocopherol (antioxidant) and attenuated by AM251 (CB1 receptor antagonist). In conclusion, based on these studies, the cannabinoid system may provide a new target for the development of drugs to treat cancer such as CRC. However, the CBs also produce seemingly unspecific cytotoxic effects, and may have negative effects on the neuronal differentiation process. This may be responsible for, at least some of, the embryotoxic effects found in ovo, but also for the cognitive and neurotoxic effects of cannabinoids in the developing and adult nervous system.

The embryonic source and chemical nature of those factor/s directing the in vivo differentiation of melanocytes from crest cells are as yet unknown. To begin to address this issue, it is important to establish exactly when and where these signa/s first exert their effects. Therefore, in the present study, overtly differentiated melanocytes containing melanin were quantitated in developing Black Australorp X New Hampshire Red chick embryos. In contrast to previous studies, it was found that embryos synthesize melanin from as early as Day 5 of development, and that at this stage, the melanocytes are predominantly dermally located. Between 5 and 8 days, the numbers of both dermal and epidermal melanocytes increase, after which the dermal melanocyte population declines rapidly while the number of epidermal melanocytes continues to increase. These findings suggest that premelanocytes do not have to be epidermally located to initiate terminal differentiation and implicate the dermis as a possible source of melanocyte inducing factor/s. The next step was to examine stages of development prior to the onset of pigment production. For this reason, tyrosinase was purified for use as antigen in the production of a polyclonal antibody. The antibody was tested for specificity by western blotting, - immunocytochemistry and immunoinhibition procedures. Lack of specificity was demonstrated, rendering it unsuitable as an antibody marker for early melanocytes. Fowl melanocytes are thought to differentiate into either eumelanosome- or pheomelanosome synthesizing cells. To test the validity of this concept, embryonic skin of the red/black cross breed were screened for possible mixed type melanocytes by electron microscopy. The melanocytes contained melanosomes with a matrix of irregularly arranged filaments amongst typical eumelanogenic melanosomes. This suggests that these chick melanocytes may synthesize both eumelanosomes and pheomelanosomes in single cells. In a further study on pure breeding New Hampshire Reds, it was found that the melanocytes contained a mixture of typical and less typical pheomelanosomes. Outer membrane indentations in the latter melanosome type suggest that tyrosinase may enter these pheomelanosomes by a mechanism related to that proposed for the melanosomes of goldfish.

Thesis (Ph. D.)--Case Western Reserve University, 2004. / [School of Medicine] Department of Neurosciences. Includes bibliographical references. Available online via OhioLINK's ETD Center.